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Emulsiflex
SET-UP: Materials: Have EtOH, 1% detergent mix (use 1g alconox in H20), diH20 (all three should be kept at machine) ready. Also ensure there is a waste beaker (is typically kept at machine also) ready to go. Bring over your WELL-ReSuspended sample (in at least 50 mL buffer) and extra sample buffer in a full bucket of ice. Ideally resuspend sample by pipetting and then use a glass douncer to make sure there are no small chunks that can clog the machine. Also have an extra beaker ready to go. Turn on high-pressure air line (in wall adjacent to machine). Gauge should read ~110psi. Turn on machine (button on back). Gently unscrew the lid for the metal sample container; make sure the sample container has been stored in EtOH and that there is some EtOH in sample container. Immerse the metal coil (midway between the rubber tubing lines) fully in an ice bucket. ****CRUCIAL: CHECK THE AIR PRESSURE GAUGE TO MAKE SURE IT IS AT 0!!! TURN KNOB TO ZERO IF IT IS NOT**** The air pressure MUST be at zero before pressing “Start” on the machine to begin pumping EVERY TIME (mid-run if pumping is stopped momentarily, as well as at beginning and end). If it is not this will seriously mess up the machine!!! If all the above is in order… OPERATING: EQUILIBRATE # Turn red button to the right until it lights up and pops out. Then press the green START button. Machine will begin pumping the EtOH out to waste. # Allow it to flow out.Then when it is almost all out (but NEVER allow to run dry), # Add ⅛ to ¼ column ofH20 1-2 times. Allow to flow through. # Pump through some sample buffer (I fill up to the “slant” line 2x here) to equilibrate machine before adding sample. ****On steps 3-4 above, it’s a good idea to turn up air regulator until the machine begins pulsing (read below for full details) in order to ensure the machine is fully cleaned and equilibrated. Just be sure there is enough liquid in container at all times so that it does NOT run dry! '''' OPERATING: LYSE SAMPLE # Pour sample into sample container. This time, WATCH the flow-through at the output tubing - transfer the tubing into your extra beaker to collect the sample as soon as the sample begins to come out. (If the sample is a GFP-tagged protein, do this transfer as soon as the flow-through it turns green. If the sample is unlabeled, wait 3-5 pumps and then transfer or watch for output liquid to become cloudy like your lysate.) # Allow one full pass (until all sample has passed thru machine into collection beaker) without further changes. **Because you do not want container to run dry this will NOT be all of material; some will be left in sample container - this is OK! # Pour all of sample back into sample container.. This time, clip the exit tubing onto the sample container to create a continuous loop. # *optional* turn the air regulator up to 20-30psi until the machine starts pulsing and watch the needle on the pressure gauge. Back the pressure down until needle is pulsing up to ~500-1000 psi. This won’t lyse your cells but will clear out any small chunks in your sample to ensure that the sample will pass through at higher psi without any problems. Run the whole sample through one time at this lower pressure. # Turn up the air regulator to about 20-30psi, or until you can hear the machine start “pulsing” and the needle on the pressure gauge begins jumping regularly. Back off the air regulator if necessary til the machine is at least at 15,000, but not quite at 20,000. **15,000 is minimum to be lysing cells but we have found it best to go to about 20,000 to get the best lysis. Air gauge can go to about 40 if needed to get this pulsing at the right intensity, but NO higher. # Cycle the sample through the machine until lysed - can take 10-15 minutes. A good gauge is when the sample goes clear instead of opaque. I would let it go at least 5 minutes longer than you think you need to for the best lysis! # Once sample is lysed, TURN AIR REGULATOR BACK DOWN TO ZERO. (You should have done this if you stopped machine AT ALL during the process!!!) # Transfer tubing back to the extra collection beaker. Collect sample. # Once nearly all sample is out, pour in a SMALL amount of sample buffer 1-3 times until nearly all of sample has been washed out. Stop collection when liquid goes clear. AT THIS POINT, stop machine (remember to turn air regulator down to zero!!). Take sample to be spun down. Come back to clean machine later. CLEANING MACHINE: # Wash through ¾ column of H20. Bring up air regulator partway through to ~30 or as needed til it starts pulsing (never higher than 40!). Pulse through for a while, then turn back down. # When almost all H20 is through, stop machine (be sure air is at 0!). Carefully remove sample container (unscrew clamp and unclip). Wash sample container with diH20 to remove bubbles and sample residue from container. # Reattach sample container. # Add ⅛ column (up to angle) of 1% detergent. Cycle, bringing the pressure up to pulse briefly again, then turn back down. # Wash with a small amount H20 several times or until bubbles are gone. # Then, run through some EtOH to kill all bacteria left. Again bring up pressure to pulse, then back down. # Stop machine. Make sure there is enough EtOH to go up to angle in sample container. Screw cap back on (gently!) to sample container. Ensure air regulator is at 0. Turn off machine. Turn off air line. Breathe. It’s okay. TROUBLESHOOTING AND IMPORTANT TIPS: # Air regulator MUST be at 0 before turning machine on to pump. NO EXCEPTIONS. # Frequently check the waste line to make sure things are OK. Trouble signs: a.If liquid is not coming out at regular pumps or appears to be blocked. b. If liquid “draws back” into tubing between pumps c.If machine begins either pumping or pulsing more quickly, or if the air pressure needle is wildly erratic In this case, there may be a blockage at one of the two valves, most often the first. In this case, probe into the sample container gently with the long wire needle. Most often if there is a blockage it can be relieved by this jostling lose of the metal bead valve inside it. If this does not help problem, call Rick - this is likely a blockage of the second valve, which is harder to reach. Will need to use high pressure air line to force sample through and you will lose any sample left in the machine. 3. When removing sample container to wash it, there is a black rubber O-ring that is crucial to form a seal between container and machine… do NOT lose it!!!